Human liver epithelial cell lines

ABSTRACT

Immortalized cell lines derived from normal adult human liver are described which express phenotypic characteristics of normal adult liver epithelial cells. The invention further provides methods of using the immortalized cells to evaluate the cytotoxicity or carcinogenicity of a compound.

RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 08/025,336,filed Mar. 3, 1993, now U.S. Pat. No. 5,665,589 which is aContinuation-In-Part of U.S. patent application Ser. No. 07/879,165,filed May 1, 1992, now U.S. Pat. No. 5,529,920 which in turn is aContinuation application of U.S. patent application Ser. No. 07/377,967filed Jul. 11, 1989, and now abandoned, which in turn was aContinuation-In-Part application of Ser. No. 07/284,368, filed Dec. 14,1988 and now abandoned, and also of Ser. No. 07/284,331, filed Dec. 14,1988 and now abandoned, all of which are hereby incorporated in theirentirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to continuous cell lines derived from normal adulthuman liver tissue. These cell lines display morphological and geneexpression characteristics consistent with parentage of normal humanhepatocytes. The cell lines are immortalized by expression of the largeT antigen (TAg) of the SV40 virus, but are not tumorigenic. As such,they provide a reproducible source of cells for studies of initiationand progression of carcinogenesis, especially chemical and viralcarcinogenesis caused by liver metabolism of non-tumorigenic precursorcompounds to genotoxic substances or by infection by oncogenic hepatitisviruses.

2. Description of the Related Art

Throughout this patent application, numerous articles of the scientificliterature are cited. Each of these references is hereby incorporated inits entirety by such citation.

Kaighn and Prince (1) described clonally-derived cultures of liver cellsfrom fetal, infant and adult human donors more than 20 years ago. Thesecultures all had limited lifespans. Their observations suggested theexistence of normal adult human liver epithelial cells that are eitherless differentiated, or are capable of undergoing retrogradedifferentiation into a form that is capable of completing a fewpopulation doublings in vitro if cultured under appropriate conditions.Cultures of rat liver epithelial cells have been established (2,3), butthese cultures, like those of Kaighn and Prince, have only limited lifespan. Recently, we described a serum-free culture medium (LCM, describedbelow) which supports extended replication of normal human liverepithelial cells (4). However, the growth potential of liver cells onthis medium was still limited, that is, no more than 12 rounds of celldivision were ever obtained in any of our cultures. All of thesecultures are not suitable for long-term studies due to their limitedlifespan.

Metabolic activation of environmental carcinogens from several chemicalclasses have been studied in human liver tissue explants or microsomesand isolated human hepatocytes. (5). Furthermore, observed animalspecies-specific differences in aflatoxin B₁ (AFB₁) and2-acetylaminofluorene metabolism indicate the need for studying humanliver or hepatocytes. However, because tissue availability is limited,individuals vary in their propensity for xenobiotic metabolism andreproducible in vitro conditions are difficult to establish, areproducible system with human liver cells for pharmaco-toxicologicalstudies has not been established.

Several groups of investigators have reported that the longevity ofcultures of human epithelial cells can be increased or in some casesmade indefinite (5,6) by transformation with the SV40 virus large Tantigen (TAg) gene. Such transformed cells may have near normalkaryotype and some of the isolates retain many of the growth anddifferentiation characteristics of their normal counterparts, includingnon-tumorigenicity. In addition, Woodworth et al. and Ledley et al.(7-9) have reported that rat hepatocytes transformed with an SV40 TAggene retained several normal hepatocyte characteristics. Unfortunately,such cultures are not useful for studies of human carcinogenesis anddrug metabolism studies, since metabolism of xenobiotic compounds can bevery different in humans and in rats.

SUMMARY OF THE INVENTION

The human liver is one of the few organs in adults that is capable ofregeneration. However, no continuously replicating culture ofnon-neoplastic, adult human hepatocytes has ever been established. Wedisclose herein the establishment of a continuous culture of normalhuman liver epithelial cells (hepatocytes) by infection of replicativecultures of such cells with a retroviral vector containing the SV40 TAggene. These cell lines (THLE cells) overcome the deficiencies ofprevious cell lines with regard to limitation of lifespan or non-humanorigin and so provide a reproducible source of cells for long-termstudies of human carcinogenesis and toxicology. The cells appear to beimmortal, that is, they have an indefinite lifespan in vitro. Cells ofthe lines described herein are non-tumorigenic and thus provide aresource for studies of processes by which cells are made tumorigenic.They are particularly valuable in the study of chemical carcinogenesis,as metabolism of non-carcinogenic precursor compounds by liver enzymesto a genotoxic compound is thought to be a major mechanism ofcarcinogenesis by chemicals. Thus the cells provide a means of screeningchemicals for carcinogenic potential by exposure of the cells to thesuspect carcinogen precursor and assay for conversion of the cells to atumorigenic phenotype. Reproducibility of such an assay depends uponhaving a reproducible cell line to carry out the tests upon. The THLEcell lines of the present invention provide such reproducible celllines.

Furthermore, the THLE cells of the present invention might prove usefulin the investigation of the control of differentiation processes. It isgenerally thought that proliferation and differentiation are opposingcellular processes (47,48). Thus, the THLE cells can be used to identifydrugs useful in the treatment of liver tumors by investigating theeffect of such drugs upon the phenotype of the THLE cells. Compoundswhich induce terminal differentiation of the THLE cells would beconsidered to be promising candidates.

The introduction of oncogenes in addition to the SV40 TAg gene can alsobe performed on the THLE cells so as to investigate the effect of theexpression of such additional oncogenes upon the tumorigenicity or otherphenotypic aspect of the THLE cells. Again, the cell lines so derivedcan be used as target in screening assays for compounds which areeffective to stop the proliferation of cells expressing these additionaloncogenes.

Finally, hepatocytes are the cell type infected by hepatitis viruses(HepA, HepB, HepC and nonA-nonB hepatitis) and also the cell typeinfected by many human parasites. Thus THLE cells provide an in vitrohost for the growth of these organisms and consequently an in vitrosystem for study of the cell biology of such infections and screening ofcompounds for efficacy in blocking or curing such infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D shows the morphology and expression of cellular markers inTHLE-2 cells; Phase-contrast micrograph showing the epithelialappearance of THLE-2 cells (A), Indirect Immunofluorescence staining forSV40 T-antigen (B) and cytokeratin 18 (C) demonstrating the presence ofboth proteins in nearly 100% of passage 5 cells, Immunoperoxidasestaining showing clonal expression of albumin in THLE-2 cells of passage5 (D). For immunofluorescence analyses, non-specific binding was blockedwith the appropriate blocking serum (1:100 dilution, 20 min.).Subsequently, antibodies (IgG) specific for albumin (1:20; AmericanQualox, La Mirada, Calif.), general cytokeratins (1:15; ICN, Costa Mesa,Calif.), cytokeratin 18 cytokeratin 19 (1:20 each; ICN, Costa Mesa,Calif.), α-fetoprotein (1:50; Zymed, San Francisco, Calif.), α₁ -antitrypsin, α₂ -macroglobulin (1:50; Chemicon, Inc., Temecula, Calif.)and SV40 T-antigen (1:5; Oncogene Science, Manhasset, N.Y.), wereincubated at room temperature (30 min to 1 hr) and developed withfluorescent secondary antibodies diluted 1:32 at room temperature (1hr.) Immunocytochemical staining for albumin was performed by incubationwith a secondary rabbit anti-mouse; DakoCorp, Santa Barbara, Calif.) andtertiary (swine anti-rabbit; DakoCorp, Santa Barbara, Calif.) antibodiesand development with horse radish peroxidase at room temperature (30 mineach incubation).

FIG. 2A-2B shows albumin secretion of THLE-2 (A) and THLE-3 (B). Whennormalized by densitometry to the simultaneously immunoprecipitatedalbumin standard of 3 ng in A and 2 μg in B approximately 300 pg/ml, 70pg/ml and 14.5 ng/ml albumin were secreted in 24 hrs by THLE-2 (Rb),THLE-2 (FL) and THLE-3 (FL), respectively.

FIG. 3A-3B shows karyotypes of THLE-2 and THLE-3. Monosomy ofchromosomes 2 and 10, a break of chromosome 1 (arrow) and a 22q+translocation leading to the marker chromosome M_(1A) characterize thenear-diploid metaphase of THLE-2 at passage 18(A). Typical SV40 Tantigen effects were also detected in THLE-3 at passage 22(B),illustrated by the monosomy of chromosomes 13 and 22 and deletions inchromosomes 2 and 8. An unidentified marker (M) chromosome is also seen.

FIG. 4A-4E shows the results of an experiment assaying metabolicactivation of carcinogens. THLE-2 cells were incubated (24 hrs) with 1.5μM ³ H-B a!p, 32 μM ³ H-AFB₁ or 50 μM DMN, respectively. Pretreatmentwith Arochlor 1254 was performed 24 hrs before carcinogen treatment. Thecells were then harvested with trypsin, suspended in lysis buffer (5-10ml), (Applied Biosystems, Foster City, Calif.), and treated withribonuclease and proteinase K (2 hrs each). Carcinogen-modified DNA wasisolated from cells by chloroform/phenol extraction (39), hydrolysed andchromatographed. BPDE-DNA adducts were identified by mixing thehydrolysed DNA bases with UV-absorbing quantities of known BPDE-DNAstandards and were fractioned on Sephadex LH20 columns and furthercharacterized by HPLC (A; ▪, Arochlor induced; o, uninduced) (24).Northern blot analysis (B) of polyA⁺ -selected mRNA showed an inductionof CYP1A1 normalized to GAPDH where Arochlor<B a!P<Arochlor+B a!P. Therelative CYPlAl/GAPDH ratios are 0.73, 10.0 and 13.1, respectively.Alkyl-DNA adduct analysis was performed by HPLC and detected by ³²P-postlabeling. Autoradiograms of the two-dimensional TLC separation ofnucleotide 32P-postlabeling assay of DNA from untreated cells (C) didnot show detectable N7-methyl deoxyguanosine adducts, but cells exposedto DMN (D) had detectable levels as demonstrated in this case at 28 fmolper μg DNA. Adducts co-eluted with UV markers of the postlabeled productto confirm adduct identity. The level of adduct was determined throughthe use of scintillation counting and calibration curves for knownversus detected molar ratios of adduct to unmodified dGp. Purified DNAfrom AFB₁ -treated cells was assayed by high performance HPLC adductpurification and detection (23). The HPLC-profiles of AFB₁ -DNA adductsat 24 hrs identified AFB₁ -fAPyr as the major product (E).Identifications are based on co-elution with authentic standards.

FIG. 5A-5B shows Northern blot analysis of phase-II enzymes. Total RNAwas isolated from THLE cells, normal human liver tissue, case 88-5,which led to the establishment of THLE-2, the hepatoblastoma cell line,HepG2, and normal human bronchial epithelial cells (NHBE). Based onethidium bromide staining (GAPDH underestimated the amount of RNA loadedfrom the liver) similar expression of epoxide hydrolase (A), GPX, SOD(B) were found in THLE cells and human liver, whereas the expression ofCAT (A) and cytochrome P450 reductase (B, NADPH-red.) was reduced.

DETAILED DESCRIPTION OF THE INVENTION

The cell lines of the present invention provide reproducible biologicalmaterials for investigations in carcinogenesis and toxicology. The cellscan be used for investigations of metabolic activation of compounds tocytotoxins or carcinogens. The cells may be used in their present state,or alternatively, exogenous genes in addition to SV40 T antigen can beintroduced into the cells. Similarly, the cell lines of the presentinvention can be infected with various viruses of interest in humandisease, such as hepatitis.

Genes of interest in studies of carcinogenesis and toxicology, forexample, might be oncogenes per se, wild-type or mutated tumorsuppressor genes or genes encoding enzymes for metabolism of xenobioticcompounds. A family of particularly interesting genes are mutants of thetumor suppressor gene p53, which have been implicated in the progressionof a variety of tumor types.

The cell lines of the present invention, either as described herein orcontaining additional exogenous genes, are useful in the screening andstudy of the mode of action of therapeutic compounds which alter geneexpression in the cells or which alter the toxicologic effects of somesecond substance.

Preferred embodiments of the invention are described by means of theexamples below. These examples are expressly meant to illustrate, ratherthan limit, the scope of the invention.

Cell proliferation is measured in a variety of the experiments describedbelow. Thus, the methods used for such measurement are set forth asgeneral methods. DNA synthesis is measured in cells inoculated at clonaldensity (100 cells/cm²). The medium is changed to fresh medium the nextday and after two additional days of incubation ³ H!-thymidine (NewEngland Nuclear) is added to the cell cultures at 0.5 μCi/ml.Twenty-four hours later the acid precipitable fraction is collected onglass fiber filters and the amount of ³ H! incorporated is quantified byscintillation counting. Alternatively, proliferation is measured bycounting the number of cells in each colony. Medium is changed to themedium in which proliferation is to be assayed one day followinginoculation at clonal density and the dishes are incubated for a further7 days. The cells are then formalin fixed and stained with crystalviolet. The number of cells per colony is determined and the populationdoublings per day is calculated as previously described (13).

For many of the experiments described, for example Southern and Northernblot analyses and metabolic experiments, cultures are grown to highdensity. Such cultures are incubated in T175 tissue culture flasks or in800 ml roller bottles and grown to a density of 3.7×10⁴ cells/cm².

EXAMPLE 1 Establishment of Continuous Cultures of Normal Adult HumanLiver Epithelial Cells (THLE cells)

i) Primary culture of normal adult liver tissue:

LCM medium (4) consists of PFMR-4 medium (Biofluids, Rockville, Md.)wherein the Ca²⁺ concentration is reduced to 0.4 mM and arginine isreplaced with 0.3 mM ornithine, supplemented with insulin (1.45 μM),transferrin (125 nM), cholera toxin (300 pM), epidermal growth factor(825 pM), hydrocortisone (0.2 μM), triiodothyronine (10 nM), retinoicacid (10 nM), phosphoethanolamine (0.5 μM), Ex-Cyte V (312 μM), bovinepituitary extract (ref. 10, 7.5 μg protein/ml), and chemically denaturedserum (10).

To make LCM medium conditioned by Hep-G2 cells (HGLCM), Hep-G2 cells(American Type Culture Collection, Rockville, Md.) are maintained inDMEM medium supplemented with 10% fetal bovine serum. Near-confluentcultures of such cells are washed twice with LCM and then maintained inLCM for 72 hours. The supernatant medium (HGLCM) is removed, sterilizedby filtration through a 0.22 μm membrane and stored under sterileconditions.

Normal liver epithelial cells are obtained by collagenase/dispaseperfusion of the left lower lobe of livers from immediate autopsy adultdonors with no clinical evidence of cancer (11). Cultures are inoculatedinto flasks that have been precoated with collagen I (Vitrogen™, CeltrixLaboratories, Palo Alto, Calif.) and incubated overnight in Waymouth'smedium containing 10% fetal bovine serum. The following day, thecultures are rinsed with phosphate buffered saline (PBS) and the mediumis changed to HGLCM.

Within 2 to 4 days of isolation of the normal cells, groups of randomlyspaced replicating cells with an epithelial-like morphology are evident.These cultures form a confluent monolayer after 10-14 days ofincubation. These normal cells can be subcultured at a 1:4 split ratiousing the same collagenase/dispase solution as is used in establishingthe primary culture to remove the cells from the surface of the culturevessel. The average lifespan of these normal liver epithelial cellcultures is 12 population doublings.

ii) Production of the SV40 TAg-expressing retrovirus

A recombinant retrovirus carrying the large T antigen gene of SV40 virusis constructed by insertion of BglI-HpaI fragment of the SV40 viral DNA(nucleotides 5235-2666) into the BamHI site of the pzipNeoSVX (12)retroviral vector, using BamHI linkers and standard recombinant DNAtechniques. The fragment of the SV40 genome employed lacks both theearly promoter and the polyadenylation site.

Infectious recombinant virus particles are made by transfecting theamphotropic packaging cell line PA317 with the ecotropic recombinantvector obtained above. Transfected cells are isolated by neomycinselection and 10 clones are isolated. The cloned PA317 cells arepropagated in DMEM medium supplemented with 10% FBS. The medium ischanged to serum-free PC-1 medium (Ventrex Laboratories, Portland, Me.)and virus is titered by infecting 8×10⁴ NIH 3T3 cells in a 60 mm dishwith various dilutions of the supernatant medium containing virus in thepresence of 8 μg/ml polybrene and counting the colonies after 10 days ofselection using 750 μg/ml of neomycin.

iii) infection of primary liver tissue culture cells

A pool of virus from 7 of the 10 clones of the transfected PA317 cellsis used to infect the primary liver tissue cultures. 8×10⁴ cells of theprimary cultures were infected with 0.1 pfu of the recombinant virus for2 hours in the presence of 8 μg/ml polybrene in PC-1 medium. After theinfection, the cultures are washed with HEPES buffered saline (HBS) andincubated in LCM medium. Infection with the recombinant virus causesvirtually all of the liver cells in the culture to undergo rapiddivision. Several cultures have been so established. Of these, THLE-2and THLE-3 are passaged as mass cultures. Initially, the THLE-2 andTHLE-3 cells undergo approximately 25 population doublings during thefirst six weeks post-infection, then growth decreases markedly. Cellsare cryopreserved at each passage during this early growth period.

The THLE-2 cell line was deposited under the terms of the BudapestTreaty at the American Type Culture Collection, 12301 Parklawn Dr.,Rockville, Md., on May 16, 1989 and assigned the accession number CRL10149. The THLE-3 cell line was deposited under the terms and conditionsof the Budapest Treat at the American Type Culture Collection on Jan.14, 1993 and was assigned the accession number CRL 11233.

THLE-2 cells from such early passage cryopreserved stocks are used todetermine the growth responses of the cells to the various supplementsof the LCM medium. Single elimination experiments show that the clonalgrowth rate of the early passage THLE-2 cells is increased by omittingthe Ex-Cyte V and cholera toxin from the LCM and also by replacing theornithine with arginine. Use of medium conditioned by THLE-2 cellsrather than by HepG2 cells further improves the growth of the THLE-2cells. Modified LCM (MLCM) medium is thus LCM reformulated by theomission of Ex-Cyte V and cholera toxin and using arginine rather thanornithine (at 0.3 mM) and adding to 30% of the volume medium conditionedby THLE-2 cells rather than by HepG2 cells.

Using MLCM to maintain the culture, THLE-2 cells have been cultured formore than 130 population doublings with no evidence of senescence. Theapparent maximal clonal growth generation time is 24 hours and theircolony forming efficiency averages 15%. THLE-3 cells were switched toMLCM at early passage and consequently this cell line never entered aquiescent stage. THLE-3 cells have been grown for more than 100population doublings. Their growth rate is 0.7 PDL/day and their colonyforming efficiency is 15%.

EXAMPLE 2 Analysis of Liver-specific Gene Expression in THLE Cells

The THLE cells were evaluated for expression of a number ofliver-specific genes at both the transcriptional and translationallevels.

Karyotype analysis is performed using techniques standard in the art.

For Southern blot analysis, cellular DNA is extracted by methods typicalin the art and digested with ClaI restriction enzyme. The DNA iselectrophoresed on a 0.7% agarose gel and transferred to Gene ScreenPlus™ (DuPont, Wilmington, Del.). Genomic DNA is analyzed for thepresence of SV40 T-antigen DNA by probing with the 1.17 kilobasepair(kb) HindIII fragment of the large T-antigen gene labelled with ³² p byuse of a nick translation kit according to the method described by themanufacturer (DuPont).

mRNA expression is assessed by both Northern blot analysis and by insitu hybridization. For Northern blotting, RNA is isolated as previouslydescribed (15) by hybridization to biotinylated oligo-dT followed bycapture of the hybridized RNA with streptavidin paramagnetic beads(Promega, Madison, Wis.). The hybridization protocol has also beenpreviously described (15).

For in situ hybridization, cells are incubated for 10 days in culturechamber slides, then washed twice with PBS (pH 7.4) and fixed for 3minutes in PBS containing 4% paraformaldehyde, 2% sucrose, 5 mM MgCl₂and 0.02% diethyl pyrocarbonate. The slides are then washed with twochanges of PBS containing 5 mM MgCl₂ and then incubated for 10 minutesin 0.1M glycine/0.2M Tris and subsequently acetylated by 10 minutesincubation in 5% acetic anhydride/0.1M triethanolamine, pH 8.0. Theslides are then washed in PBS and prehybridized in 50% formamide, 2×SSC, 10 nM dithiothreitol (DTT) at 52° C. for 10 minutes. Hybridizationis performed at 50° C. using cRNA probes or 42° C. using cDNA probes in50% formamide, 2× SSC, 0.1M DTT, 1 mg/ml tRNA, 10 mg/ml sonicated salmonsperm DNA, 2 mg/ml bovine serum albumin (BSA) and 6×10⁴ cpm/μl of probe.Following hybridization, slides probed with cRNA are rinsed in 50%formamide, 5× SSC, at 50° C. for one hour, then RNAse digested asdescribed by Maier et al. (16). The slides are then washed at 45° C. in50% formamide, 2× SSC for 30 minutes and finally in 2× SSC for 30minutes at 45° C. Slides probed with CDNA probes are rinsed in 50%formamide, 2× SSC at 37° C. for 30 minutes, then in 50% formamide, 1×SSC at room temperature and finally in 1× SSC at room temperature.Hybridized slides are then autoradiographed using NBT2 emulsion (EastmanKodak), exposed at 4° C. for 7 to 10 days and developed with Kodak D19developer, then counterstained with hematoxylin and eosin, dehydratedand mounted with Permount.

CDNA probes are labelled by nick translation using ³⁵ S dCTP assubstrate. The average length of such probes is 0.2 kb. Riboprobes(cRNA) are prepared by the transcription protocol of Melton et al. (17)using ³⁵ S UTP as the labelled nucleotide. The transcripts are thenpartially hydrolyzed with alkali so that most of the label is infragments 100-200 nucleotides long.

The presence of epoxide hydrolase is determined in the cell lines byhybridization with a human gene probe; either the SmaI-XhoI (0.4 kb) orthe NcoI-NspI (0.9 kb) fragments of the plasmid R60 (OxfordBiochemicals, Oxford, Mich. 48051). NADPH cytochrome p450 reductaseprobe is derived from plasmid hp450 (F. Gonzales, National CancerInstitute, National Institutes of Health, Bethesda, Md.) by EcoRIdigestion and isolation of the 2.4 kb fragment. Superoxide dismutase(SOD) expression is determined by hybridization to a 0.45 kbEcoRI-HindIII fragment of the human CDNA obtained from the plasmidsp65/SOD (18). Glutathione peroxidase is analyzed by use of a 0.8 kbEcoRI fragment of the human CDNA obtained from the plasmid pSPT19/GPX(19) Expression of Glutathione-S-transferase πi, a and A are assessed byhybridization with the 0.73 kb EcoRI fragment of plasmid pGEM4/GSTπi (J.A. Moxow, National Cancer Institute, National Institutes of Health,Bethesda, Md.), to the 0.7 kb EcoRI fragment of plasmid pGST2-PvuII (20)and to the 0.67 kb PstI-EcoRI fragment of the plasmid pGST-T-Nco (giftof P. G. Board, Australian National University), respectively. Probe foralbumin mRNA is isolated from plasmid B44 (21). The 0.73 kb insert cDNAof B44 is subcloned in pGEM4 (Promega, Madison, Wis.) between the PstIand HindIII sites. For detection of catalase mRNA, nick-translated probeis prepared from the 1.25 kb EcoRI-HindIII fragment of a plasmidcontaining the HindIII-PvuII fragment of the human catalase CDNA (22).Probes for cytochrome p450 isoenzymes correspond to the 1.0 kb 3' EcoRI1A2, 1.0 kb 3' EcoRI 1A1, 1.3 kb 3' BamHI-EcoRI IIA3, the 3+ 1.6 kbBamHI-EcoRI IIE1, the 1.1 kb 3' EcoRI IIIA4, the 0.8 kb 5' EcoRI IIB1CDNA fragment or the entire 1.6 kb IID6 cDNA isolated by EcoRI digestionof plasmids provided by F. Gonzales (National Cancer Institute, NationalInstitutes of Health, Bethesda, Md.).

For immunocytochemistry, cells are grown to near confluence on glasschamber slides (Lab-Tek) and rinsed in phosphate-buffered saline (PBS).The cells are fixed by immersion of the slides in phosphate buffercontaining paraformaldehyde (for albumin staining) or 100% methanol at4° C. (for cytokeratin and TAg staining). The slides are then rinsed inPBS and the appropriate blocking serum (1:100 dilution) is placed oneach slide for 20 minutes. The primary antibodies (IgG) against albumin(1:20 dilution, ICN, Costa Mesa, Calif.), general cytokeratins (1:15dilution, ICN), cytokeratin 18 and cytokeratin 19 (each at 1:20dilution, ICN) and SV40 T-antigen (1:5 dilution, Oncogene Science,Manhasset, N.Y.) are applied to the slides and incubated for 30 minutesto 1 hour at room temperature. The slides are rinsed in PBS to removeunbound antibody. For immunofluorescence studies, fluoresceinisothiocyanate or tetramethyl rhodamine isothiocyanate labelledsecondary antibodies are placed on the slides for 1 hour at roomtemperature. For staining with horseradish peroxidase (HRP) the slidesare incubated with swine anti-mouse secondary antibody for 30 minutes atroom temperature, rinsed in PBS and then incubated with HRP-linkedrabbit anti-swine tertiary antibody under the same conditions. HRP isdetected by turnover of a benzidine substrate.

For negative control experiments, 3T6 mouse fibroblast cells were used.HepG2 cells were used as a positive control cell line.

Albumin secretion from the THLE cells was assayed by Western blotanalysis of immunoprecipitated albumin. Cell supernatants (10 ml from 72hr cultures of approximately 0.6×10⁶ cells/ml in flasks or 1.2×10⁶cells/ml in roller bottles, the cells were switched to LCM withoutconditioned medium 24 hours before the assay) were adjusted to the saltand detergent concentration of RIPA (1×) (40), the albuminimmunoprecipitated (4° C.: 1 hr) with goat anti-human albumin (Dako,Santa Barbara, Calif.) and subjected to Western blot analysis andquantitative densitometry (FIG. 2). Albumin was isolated from cellsgrown in serum free medium for 24 hours in roller bottles (Rb) or flasks(F1) (0.6×10⁶ /ml respectively; 72 hrs) with 10 μl of goat anti-humanalbumin followed by extraction (1 hr) with protein A sepharose (Zymed,South San Francisco, Calif.). The immunocomplexes were washed twice withRIPA buffer, once in a mixture of equal volumes of RIPA and TNE (0.15MNaCl; 0.05M Tris-Hcl, pH 7.5; 1 mM EDTA) and once in TNE. The albuminprotein was eluted in a sample buffer (200 μl, 0.06M Tris-HCl, ph 6.8,2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.002% bromophenol blue),electrophoresed on a 7.5 SDS polyacrylamide gels and transferred to anitrocellulose membrane. The membrane was blocked for nonspecificbinding at room temperature (1 hr) with non-fat milk (5%) diluted inTBST (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) beforehybridizing with a rabbit anti-human albumin antibody (Dako, SantaBarbara, Calif.) diluted 1:800 in TBST including 5% non-fat milk.Subsequently, the membrane was washed three times in TBST (10 min),incubated at room temperature with a swine anti-rabbit biotinylatedantibody diluted 1:2000 in TBST (30 min), washed again as above andincubated at room temperature in streptavidin alkaline phosphatase inTBST (30 min) which generated a color reaction in the presence of thechromogen (ABComplex, Dako, Santa Barbara, Calif.).

Karyotype analysis of THLE-2 and THLE-3 cells shows that both lines arehypodiploid (aneuploid) with most karyotypes being near diploid. Eachcell line's karyotype is distinctive; neither is completely normal. Bothexhibit structural alterations such as chromatid breaks, deletions andacentric fragments. Both THLE-2 and THLE-3 have been tested for tumorformation by subcutaneous injection into athymic nude mice; no tumorshave arisen after 12 months following injection of the cells.

Southern blot analysis of DNA from the THLE-2 cells shows that thesecells contain a single copy of the SV40 T-antigen gene per haploidgenome. Cultures of the SV40 TAg immortalized cells are passaged as masscultures. Immunocytochemical analysis of both THLE-2 and THLE-3 cellsbetween the 3rd and 5th passage shows that all of the cells of bothlines express TAg in their nuclei.

At early passage (passage 3), both THLE-2 and THLE-3 cells demonstrateimmunocytochemical evidence of expression of cytokeratin 18, but notcytokeratin 19. The early passage cells also stain positively foralbumin. α-fetoprotein is not detectable by immunostaining. In situhybridization to mRNA demonstrate confirms the positive expression ofalbumin and lack of expression of α-fetoprotein. In situ hybridizationalso detects mRNA encoding transferrin, α2-macroglobulin andα-1-antitrypsin.

Re-examination of cytokeratin expression by immunostaining at passage 10shows that both cytokeratin 18 and cytokeratin 19 are expressed at thelater passage in both cell lines. Albumin expression in later passagecells (passage 12) is dependent upon culture conditions. Growth underconditions favoring rapid proliferation results in lower albuminexpression. Growth under conditions which slow proliferation, such asroller bottle culture or plating on collagen or MATRIGEL™ surfacesresults in increased albumin expression. Albumin secretion by THLE-2cells in roller culture was approximately 300 pg/ml of culture medium.In flask culture, THLE-2 and THLE-3 cells produced 70 pg/ml and 14.5ng/ml of albumin, respectively. Immunocytochemical analysis of albuminexpression shows that albumin is readily detected in early passageTHLE-2 an THLE-3 cells. Islands of cells staining for albumin weresurrounded by clusters of less intensely staining cells, indicating thepresence of different clonal types in the culture at low PDL.

THLE-2 cells have been evaluated for expression of a number ofhepatocyte-specific markers. THLE-2 cells express cytokeratin 18 and notcytokeratin 19 at early passage, while at later passage, expression ofboth of these cytokeratins is observed. Since cytokeratin 19 is notnormally expressed in vivo in hepatocytes, but is expressed in bile ductcells, this is an indication that the THLE cell lines eitherdedifferentiate toward a more primitive, less committed cell type duringpassage or that a stem cell type gains a selective advantage in theculture. Evidence that bile ductal cells and hepatocytes arise from acommon stem cell has recently been shown by others (44).

Cells for γ-GT staining were plated onto coated glass chamber slides,washed in PBS, fixed in ice-cold acetone (2 min), and stored at -20° C.until used. Enzyme histochemical reaction for γ-GT was performed asdescribed (41). The HepG2 cell line was used as a positive control andthe hamster embryonic cell line 3T6 as a negative control. γ-GT wasweakly detected by immunocytochemistry in some colonies of THLE-2 andTHLE-3, as well as in primary cultures prior to viral transformation.3T6 cells were negative, whereas HepG2 cells uniformly exhibited highlevels of the enzyme.

For Factor VIII analysis, cells were fixed in ice-cold acetone (2 min)and incubated with a mouse monoclonal antibody to human factor VIII (45min; Zymed) at room temperature. Primary cultures of human umbilicalcord endothelial cells were used as a positive control. Factor VIIIexpression was not detected at early or late passage in THLE cells.

Expression of catalase, superoxide dismutase and glutathione peroxidaseis evaluated in THLE-2 cells by Northern blot analysis. The cells areshown to express MRNA for each of these proteins.

When evaluated for mRNA expression, early passage THLE-2 cells are foundto express messages for albumin, transferrin, α-1 antitrypsin,α2-macroglobulin, catalase, superoxide dismutase and glutathioneperoxidase. α-fetoprotein expression is not detectable at either themRNA or protein level. α-fetoprotein is not normally expressed by maturehepatocytes, but is secreted by regenerating liver and often byhepatocellular carcinoma. Thus, THLE-2 cells demonstrate a pattern ofgene expression similar to that of normal hepatocytes in vivo. Thispattern of gene expression indicates that cultures of THLE-2 cells (andprobably THLE-3 cells, though they are not as well characterized) aremostly a dividing population of liver cells which maintain at least apartially differentiated phenotype.

Early passage THLE-2 and THLE-3 cells formed colonies with mixed abilityto secrete albumin. We hypothesize that these cells constitutededifferentiated hepatocytes that have varying ability to expressalbumin or arose from liver stem cells differentiated to cells withhepatocyte characteristics. In rats treated with hepatic carcinogens ortoxic compounds, oval cells that are much smaller than parenchymalhepatocytes or nodular cells are observed (42-45). Oval cells candifferentiate to liver parenchymal cells under particular conditions invivo (43,45,46) suggesting that these cells may be stem cells with thepotential of being neoplastically transformed to cholangiocellular, aswell as hepatocellular carcinomas (44). Rat oval cells are characterizedby the expression of phenotypic markers such as albumin, cytokeratin 18and 19, γ-GT, α-fetoprotein and glutathione-S-transferase pi, whereas6-glucose phosphatase activity is only weakly positive (43,45). THLEcells have an epithelial morphology; early passage cells secretedalbumin, expressed cytokeratin 18, transferrin, α₁ -antitrypsin,α-macroglobulin, GST (FIGS. 1 and 2), and very low levels of γ-GT. Theywere uniformly negative for α-fetoprotein and factor VIII. Therefore,THLE cells represent a population with a differentiation grade betweenoval cells and hepatocytes. The possibility that the THLE cells arederived from hepatocyte precursors such as oval cells cannot be ruledout. However, the fact that cytokeratin 18 is expressed andα-fetoprotein is absent in a very early stage of their establishmentindicates a derivation from differentiated hepatocytes rather than ovalcells. The appearance of cytokeratin 19 and the decrease in albuminsecretion at later passages suggests that the cells dedifferentiate inculture, a process often seen as a consequence of transformation (47).In the in vitro model of normal liver epithelial cells described here,dedifferentiation is reversible because albumin expression can beinduced in roller bottles and by growing cells on extracellular matricesor in tridimensional aggregates.

A second consequence of dedifferentiation of hepatocytes is the loss ofdrug metabolizing enzymes including cytochrome P-450 and associatedmixed-function oxidases (48). Culture conditions such as extracellularmatrices (49-51), co-culture systems (43,52) and hormone supplementation(7,53,54) have been reported to positively influence differentiatedfunctions including phase I and II enzyme activities of primaryhepatocytes (50,55). Although SV-40 immortalized rat liver cell lineshave not been extensively characterized for their metabolic potential,maintenance and/or inducibility of CYP2B and CYPIA, NADPH dytochromeP450 reductase, glutathione S-transferases andUDP-glucuronyltransferases with levels higher than in human or rathepatoma cell lines have been reported (48). THLE cells expressed mRNAsof phase II enzymes such as epoxide hydrolase, CAT, GPD, SOD and GSTs atlevels comparable to human liver. GST pi and oc mRNAs are the dominantforms observed in both THLE cells and human liver, respectively. NADPHcytochrome P450 reductase was maintained but at a lower steady stateMRNA level than in human liver.

EXAMPLE 3 Carcinogen Metabolism by THLE cell lines, DNA Adduct Formation

Metabolism of three carcinogen precursor compounds to form DNA adductswas evaluated in the THLE cell lines. Benzo- a!-pyrene was used as aprototype compound of the polycyclic aromatic hydrocarbon class ofcarcinogen precursors. Similarly dimethylnitrosamine served as theprototype for the N-nitrosoamine class of carcinogen precursors andaflatoxin B₁ serves the prototype for the microtoxins, which arecompounds made by microorganisms that have been shown to be metabolizedby the mammalian liver to carcinogenic compounds.

For some experiments, one-half of the cultures are maintained in MLCM.The remainder of the cultures are treated with 10 μg/ml of Arochlor 1254(NCI Chemical Repository, Kansas City, Mo.) twenty-four hours prior toincubation with either tritiated benzo-a-pyrene (B a!P) or tritiatedaflatoxin B₁ (AFB₁). In other experiments, the experimental cultures aretreated with ³ H!-B a!P, ³ H!-AFB1 or ³ H!-dimethylnitrosamine (DMN) for24 hours without prior Arochlor treatment. Cells are isolated bytrypsinization and pelleted by centrifugation at 200×g for 5 minutes.The supernatant is discarded and the cell pellet is resuspended in 5-10ml of lysis buffer (Applied Biosystems, Foster City, Calif.). The lysissolution is incubated in RNAse for 2 hours, followed by a 2 hourtreatment with proteinase K. DNA is purified from the lysis mixture byethanol precipitation from the aqueous solution followingchloroform/phenol extraction.

Purified AFB1-adducted DNA, re-dissolved in water, is adjusted of 0.15NHCl and incubated for 15 minutes at 90°-95° C., as described previously(Groopman et al. (23). This procedure releases greater than 95.5% of thecovalently bound aflatoxins from the modified DNA. The hydrolysates arerapidly cooled on ice and adjusted to pH 5.3 with 1M ammonium formate.High performance liquid chromatography (HPLC) grade methanol is added toa final concentration of 5% and the samples are placed on a C-18 Sep-Pakcolumn (Waters Assoc., Milford, Mass.), washed with 5% methanol in waterto remove unhydrolyzed DNA and then eluted with 80% methanol in water.Subsequently, the solvent is removed from the eluate by rotaryevaporation under reduced pressure to a 200-300 μl sample size forstandard HPLC analysis (23).

DMN-DNA adduct analysis is performed with a combined HPLC and ³²P-postlabelling assay as previously reported (24). Briefly, 100 μg ofDNA is enzymatically digested to 3'-monophosphate nucleotides and thenpurified with ion-pair, reverse phase HPLC. Fractions containingN7-methyldeoxyguanosine (N7medGp) are mixed with deoxyguanosine (dGp) asan internal standard in the presence of polynucleotide kinase and32P-gamma-ATP. Radioactive orthophosphates are thereby transferred tounmodified and adducted nucleotides. These are resolved and quantitatedusing two-dimensional thin layer chromatography, autoradiography andscintillation counting.

Analysis of B a!P-DNA (BPDE-DNA) adducts is performed as previouslydescribed. Briefly, the DNA is hydrolyzed with DNAse I, alkalinephosphatase and phosphodiesterase and then mixed with UV-absorbingquantities of authentic BPDE-DNA adducts. The mixtures are applied toSephadex LH20 columns (90 cm×5 cm, Pharmacia LKB, Piscataway, N.J.) andeluted with water-methanol gradients (30-100% over 1 liter). Fractions(5 ml) are analyzed for fluorescence emission (excitation 340 nm,emission 400 nm) and portions (1 ml) of each are subjected to liquidscintillation counting. Fractions containing radioactive and fluorescentmaterials are further characterized by HPLC for confirmation of adductidentity.

THLE-2 cells exposed to dimethylnitrosamine, aflatoxin B1 and benzo-a!-pyrene show a dose-dependent cytotoxicity, suggesting that thesecells have the ability to metabolize these compounds to genotoxicmetabolites. Therefore, the formation of DNA adducts by thesemetabolites is investigated using THLE-2 and THLE-3 cells cultured inflasks or in roller bottles. The results of such studies are summarizedin Table 2 and FIG. 5. Roller bottle cultures of both THLE-2 and THLE-3cells

                  TABLE 2                                                         ______________________________________                                        Carcinogen-DNA adducts formed in THLE-2 cells                                 compound     uninduced  Arochlor-induced                                      ______________________________________                                        B .sup.a !P  1.5 ±                                                                              0.1.sup.b                                                                            4.9 ± 2.1                                      AFB.sub.1    2.5 ±                                                                              0.9    1.6 ± 0.4                                      DMN          30.4 ±                                                                             3.9    3.4 ± 0.1                                      ______________________________________                                         .sup.a DNA isolated from cells treated with the vehicle only were negativ     for adducts of the carcinogens examined.                                      .sup.b mean ± standard deviation; fmol per μg DNA; each value given     is calculated from two separate experiments, each having one observation.

demonstrate higher levels of adduct formation than the level observed incells grown in flasks. No metabolism of AFB1 or B a!P by THLE-2 cells isdetectable when the cultures are maintained in flasks. However, both ofthese carcinogens are readily metabolized by cells incubated in rollerbottle cultures. Metabolism of AFB1 by THLE-3 cells is similar to thatobserved in THLE-2 cells. However, metabolism of B a!P to DNA-bindingelectrophiles by THLE-3 is independent of the vessel in which theculture is maintained.

The p450-inducing agent Arochlor significantly increases the rate ofadduct formation by B a!P, but has no effect on AFB1 adduct formation.The increase in DNA adduct formation by Arochlor treatment is paralleledby induction of cytochrome p450 1A1 mRNA (FIG. 5). In cells not treatedwith Arochlor, cytochrome p450 1A1, and other p450 enzymes are notdetectable under the culture conditions described. The metabolism of theparent B a!P compound to the reactive electophile involves the action ofeither of two cytochrome p450 enzymes (P4501A1 and P450IIIA4) as well asthe phase II biotransformation enzyme epoxide hydrolase (14). The amountof B a!P dihydrodiol epoxide adduct found following exposure of THLE-2cells to B a!P indicates that at least one of the P450 enzymes as wellas epoxide hydrolase are active and regulated by Arochlor 1254. SinceArochlor 1254 has been shown to induce the enzymatic activity of severalforms of P450 in vivo (25), both of the THLE-2 and THLE-3 cell linesrespond to such treatment in a physiologically relevant manner.

The results of the carcinogen metabolism studies are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Carcinogen-DNA adducts formed in THLE-2 cells                                 compound     uninduced  Arochlor-induced                                      ______________________________________                                        B .sup.a !P  1.5 ±                                                                              0.1.sup.b                                                                            4.9 ± 2.1                                      AFB.sub.1    2.5 ±                                                                              0.9    1.6 ± 0.4                                      DMN          30.4 ±                                                                             3.9    3.4 ± 0.1                                      ______________________________________                                         .sup.a DNA isolated from cells treated with the vehicle only were negativ     for adducts of the carcinogens examined.                                      .sup.b mean ± standard deviation; fmol per μg DNA; each value given     is calculated from two separate experiments, each having one observation.

The invention being thus described, various modifications of thematerials or methods set forth will be apparent to one of skill in theart. Such variations in the scope of the invention are to be understoodas encompassed by the invention as claimed below.

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What is claimed is:
 1. A method for evaluating the cytotoxicity of acompound which comprises:1) providing cells isolated from normal adulthuman liver tissue which have the following characteristics:a)immortalized by transformation with a virus selected from the groupconsisting of SV40 virus, adenovirus, and papilloma virus; b) capacitywhen contacted with a compound to metabolize the compound to a product,which forms an adduct with DNA; and c) non-tumorigenic; 2) culturingone-half of cells of step (1) in a medium containing said compound to beevaluated; 3) culturing the remainder of the cells of step (1) in amedium lacking said compound to be evaluated; 4) measuring the survivalof the cells in step (2); 5) measuring the survival of the cells in step(3); 6) comparing the results of steps (4) and (5).
 2. A method forevaluating the cytotoxicity of a compound which comprises:1) providingcells isolated from normal adult human liver tissue which have thefollowing characteristics:a) immortalized by transformation with a virusselected from the group consisting of SV40 virus, adenovirus, andpapilloma virus; b) capacity when contacted with a compound tometabolize the compound to a product, which forms an adduct with DNA;and c) non-tumorigenic; 2) incubating one-half of cells of step (1) in amedium which lacks said compound; 3) incubating the remainder of thecells of step (1) in a medium which contains said compound; 4) isolatingthe DNA from cells of step (2); 5) measuring the formation of adducts tothe DNA isolated in step (4); 6) isolating the DNA from cells of step(3); 7) measuring the formation of adducts to the DNA isolated in step(6); and 8) comparing the amount of adduct formation measured in step(5) with that measured in step (7).
 3. A method for screening a compoundfor potential carcinogenicity, which comprises:1) providing cellsisolated from normal adult human liver tissue which have the followingcharacteristics:a) immortalized by transformation with a virus selectedfrom the group consisting of SV40 virus, adenovirus, and papillomavirus; b) capacity when contacted with a compound to metabolize thecompound to a product, which forms an adduct with DNA; and c)non-tumorigenic; 2) incubating one-half of the cells of step (1) in amedium which lacks said compound, 3) incubating the remainder of thecells of step (1) in a medium which contains said compound; 4) measuringthe expression of at least one cytochrome P450 enzyme in the cells ofstep (2); 5) measuring the expression of at least one cytochrome P450enzyme in the cells of step (3); 6) comparing the amount of cytochromeP450 expression measured in step (4) with the amount measured in step(5).
 4. A method as recited in claim 2, which further comprises steps:9)measuring also the expression of epoxide hydrolase in the cells of step(2); 10) measuring also the expression of epoxide hydrolase in the cellsof step (3); 11) comparing the amount of epoxide hydrolase expressionmeasured in step (9) with the amount measured in step (10).